A p53 is a tumor suppressor gene playing a key role in a system that maintains cell genome stability. This normally unstable nuclear protein stabilizes after DNA damage and causes G1 arrest, allowing the cell to repair the lesions before the onset of DNA synthesis. p53 is involved in programmed cell death since its function is required for cell senescence and for initiation of apoptosis after DNA damage. The p53 gene is functionally inactivated in a wide variety of human neoplasia. Components of the p53 signaling pathway acting either up- or downstream of p53 are mostly unknown. Suppression of genes encoding such components would likely induce similar alterations of cell phenotype as does the inactivation of p53 and, therefore, can be used as a basis for their identification. A general methodology for production of genetic suppressor elements (GSEs) has been recently developed and applied for identification of new genes whose inactivation is associated with different selectable phenotypes. It is based on the isolation of biologically active clones from retroviral libraries carrying random fragments of the target genes (RFRLs). GSEs are short cDNA fragments encoding either inhibitory antisense RNAs or dominant negative truncated proteins that act against genes from which they were derived. This approach has been used in mammalian cells to isolate GSEs for human topoisomerase II that cause resistance to cytotoxic drugs poisoning topoisomerase II. In an extension of this methodology, large RFRLs (>10/7 recombinant clones each) carrying random fragments of normalized (uniform abundance) cDNAs from NIH 3T3 and HeLa cells have been generated. These libraries have been already used to isolate several GSEs, inducing drug resistance or cell transformation and derived from previously unknown genes. Under the present proposal, RFRLs prepared from fragments of either rat p53 cDNA or normalized cellular cDNAs will be applied for the isolation of GSEs interfering with the p53 signaling pathway. Anti-p53 GSEs capable of increasing the life span of rat embryo fibroblasts will be isolated from the p53 RFRL. Determination of position of sense-oriented elements in p53 cDNA will probably lead to the identification of additional functional domains of p53. Selection and study of anti-p53 GSEs from cellular cDNA RFRLs will allow identification of presently unknown components of the p53 pathway. The selection will be carried out on mouse and human cells with inducible p53 expression: such cells undergo growth arrest and apoptosis after p53 induction. GSEs capable of inducing cell resistance to the p53-related effects will be isolated and used as probes to clone full-length cDNA for the corresponding genes. To study the function of these putative tumor suppressor genes, cell phenotypes associated with their inhibition or overexpression will be analyzed (interaction with p53, place in p53 pathway, involvement in cell senescence and aging, expression and role in cell reaction to DNA damage, etc.). Expression of the GSE-corresponding genes will be studied in normal cells and tumor cell lines to determine their possible involvement in neoplastic transformation.